This research is designed to explore the production of podophyllotoxin (PPT), an important natural product used in the semi-synthesis of etoposide, etopophos and teniposide for cancer treatment. The availability of PPT is becoming less assured and more expensive due to over-harvesting of the non-cultivable source plants and lack of a cost-efficient synthetic process. We have identified two fungi endophytic in Podophyllum peltatum, a North American source of the compound that are capable of biosynthesis of PPT. We have increased PPT production through fermentation optimization studies, but the rate of biosynthesis is still insufficient to meet commercial needs. The slow growth of these endophytic fungi is another significant barrier to increased production. We will conduct research that is focused on the determination of the biosynthesis steps to PPT in these fungi and identification of the genes involved in the biosynthesis of PPT in these fungi. The complete elucidation of the PPT biosynthetic pathway will have significant short-term benefits in its applicability to other PPT-producing organisms, as this has not been accomplished in any organism. The long-term benefits of the research will be to assure the supply of PPT through production on an industrial scale through fermentation, making this valuable precursor compound available for cancer therapy and continued drug development studies. PUBLIC HEALTH RELEVANCE: Shortages in podophyllotoxin for anti-cancer drug delivery and development are projected due to poor life histories of the source plants and lack of an economic route to synthesis. Endophytic fungi that produce podophyllotoxin provide a new source for the supply of this drug precursor. This project will lead to a better understanding of podophyllotoxin biosynthesis in the fungus and ultimately will lead to a sustainable and controlled supply of the compound for patient treatment and further drug development.